Promiscuity of an unrelated anthrol reductase ofTalaromyces islandicusWF-38-12

Article abstract of DOI:10.1039/d0cy02148b

An anthrol reductase ofTalaromyces islandicusWF-38-12 (ARti-2) from an unrelated biosynthetic gene cluster (BGC) has been identified and characterized. It catalyses the NADPH-dependent reduction of anthrols (hydroanthraquinones), estrone and a naphthol with high stereo- and regioselectivity. The role of ARti-2, theCRG89872.1gene of the same BGC and non-enzymatic oxidation in the biosynthesis of (?)-flavoskyrin has been proposed.

Full text of DOI:10.1039/d0cy02148b

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Promiscuity of an unrelated anthrol reductase of
Talaromyces islandicus WF-38-12†
Cite this: Catal. Sci. Technol., 2021,
1, 474
1
ab
a
a
Shailesh Kumar Singh,
‡
Anshul Rajput, ‡ Arijit De,
b
a
Received 6th November 2020,
Accepted 31st December 2020
Tapati Chakraborti and Syed Masood Husain
*
DOI: 10.1039/d0cy02148b
rsc.li/catalysis
An anthrol reductase of Talaromyces islandicus WF-38-12 (ARti-
which have been identified only recently, include MdpC of
5
7
2
) from an unrelated biosynthetic gene cluster (BGC) has been
identified and characterized. It catalyses the NADPH-dependent
reduction of anthrols (hydroanthraquinones), estrone and
naphthol with high stereo- and regioselectivity. The role of ARti-
, the CRG89872.1 gene of the same BGC and non-enzymatic
Aspergillus nidulans, AflM of A. parasiticus, 17β-HSD of
6
Curvularia lunata, and ARti of Talaromyces islandicus
2
a
(previously Penicillium islandicum). In a short span of time,
the ARs have been identified for their role in the biosynthesis
2
of chrysophanol (11), cladofulvin, monodictyphenones and
8–10
oxidation in the biosynthesis of (−)-flavoskyrin has been
proposed.
xanthone derivatives.
Furthermore, ARs might have a crucial role in the
1
1
biosynthesis of dimeric bisanthraquinoids
such as (−)-
The superfamily of short-chain dehydrogenases/reductases
flavoskyrin (30), (−)-rugulosin (31) and related metabolites
isolated from P. islandicum. Hence, apart from the recently
(SDRs) consists of enzymes with diverse functions and uses
12
NAD(P)(H) as a cofactor to catalyse oxidation/reduction
identified ARti, we expect the presence of more such
enzyme(s) which might be involved in the biosynthesis of 30,
1
reactions in a highly stereo- and regioselective manner.
Among these, naphthol and anthrol reductases (NRs & ARs)
can catalyse the NADPH-dependent reduction of polyhydroxy
31 & related natural products.
The pairwise sequence alignment of 17β-HSDcl (GenBank
2
aromatics in general. Phylogenetic studies reveal that both
AAD12052.1) against the T. islandicus (taxid: 28573) genome
yielded two significant hits: CRG89873.1 (83.71% identity, 6 ×
have emerged from keto reductases such as glucose
dehydrogenase (GDH) & alcohol dehydrogenase (ADH), and
−167
−156
10
e-value) and CRG86682.1 (79.55% identity, 4 × 10
catalyse the reduction of ketones (1) to secondary alcohols
e-value) but only the latter one is characterized as an anthrol
3
(
2) (Fig. 1). As a physiological function, NRs such as the
1
,3,6,8-tetrahydroxy naphthalene reductase (T
trihydroxy naphthalene reductase (T HNR) of Magnaporthe
grisea catalyse the NADPH-dependent reduction of naphthols
such as T HN (3) & T HN (4) to (R)-vermelone (5) and (R)-
4
HNR) & 1,6,8-
3
3
4
4
scytalone (6), respectively (Fig. 1). Likewise, ARs reduce an
unknown tautomer of an anthrol (7) formed in situ by the
2 2 4
reduction of emodin (9) in the presence of Na S O to give a
putative biosynthetic intermediate, (R)-3,8,9,10-tetrahydroxy-6-
2
,5,6
methyl-3,4-dihydroanthracen-1(2H)-one (8) (Fig. 1).
ARs,
a
Molecular Synthesis and Drug Discovery Unit, Centre of Biomedical Research,
SGPGIMS Campus, Raebareli Road, Lucknow-226014, India.
E-mail: smhusain@cbmr.res.in, smhusain.cbmr@gmail.com
b
Department of Biochemistry and Biophysics, University of Kalyani, Nadia-741235,
West Bengal, India
†
Electronic supplementary information (ESI) available: Experimental methods,
supplementary figures and tables. See DOI: 10.1039/d0cy02148b
Fig. 1 Phylogenetic tree depicting the functional diversity of NAD(P)-
‡
These authors contributed equally to this work.
dependent oxidoreductases.
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reductase (ARti) based on its conserved domains and
methyl-3,4-dihydroanthracen-1(2H)-one (8) obtained in 74%
yield. Herein, the absolute configuration was assigned using
CD spectroscopy. Chiral HPLC confirms the enantiopurity of
2
biosynthetic gene cluster (BGC) analysis.
All other
alignments were less significant due to low sequence identity
<51%). conserved domain search (CDD v3.18) of
CRG89873.1 at NCBI predicts it as a classical SDR with a
characteristic T HN/T HN reductase domain belonging to
1
5
(
A
(R)-8 to be >99% ee (see the ESI,† pages S10 and S21).
Along with 8, we also obtained chrysophanol (11) as a side
3
4
product, which is known to be formed by non-enzymatic
5
naphthol/anthrol reductases. More specifically CRG89873.1 is
proposed as an anthrol reductase (ARti-2) as inferred from
the phylogenetic tree (see the ESI,† Fig. S2). Also, its multiple
sequence alignment with known ARs (MdpC, AflM, 17β-
HSDcl & ARti) shows a common NAD(P)-binding site as well
as a conserved YXXXK active site motif (Fig. S1†). Serine (S-
oxidation, followed by dehydration. We also performed the
above transformation by taking NADH as
a cofactor.
However, no conversion to the product was observed
indicating NADPH to be the accepted cofactor for ARti-2.
Also, no product was obtained when the transformation was
2 2 4
performed in the absence of Na S O , indicating that ARti-2
1
22) & asparagine (N-148) are also present upstream of
does not accept emodin (9) as a direct substrate.
tyrosine (Y-162) & lysine (K-166) residues forming a catalytic
tetrad. Further, fungiSmash analysis of the T. islandicus
Next, we sought to determine the thermal stability &
optimum pH of ARti-2. For this, the melting temperature
1
3
genome predicts the corresponding gene of ARti-2 as part of
a putative cluster of unknown type (see the ESI,† Table S1).
(T ) was determined by recording the ellipticity of ARti-2 at
m
1
4
222 nm as a function of temperature (20–85 °C) and was
found to be 43 °C (Fig. 2a). Furthermore, the ellipticity versus
temperature plot gave us the idea to choose the optimum
range of temperature for enzymatic transformations, i.e., 20–
30 °C at which ARti-2 remains in native conformation. Also,
the optimal pH of ARti-2 was determined by performing the
chemoenzymatic reduction of 9 at pH ranging from 5.5 to
8.0. Emodin was incubated with ARti-2 and NADPH
However, ARti-2 doesn't belong to any known BGC, and in
turn, we failed to deduce its physiological functions based on
the location. In contrast, all the previously characterized ARs:
ARti, MdpC, AflM and 17β-HSDcl from different fungi were
predicted to be part of a BGC that might be involved in the
synthesis and modifications of anthraquinones like emodin
2
,5–7
(9) to chrysophanol (11).
Hence, the existence of a second
+
AR in T. islandicus at an unrelated locus raises questions
about its catalytic function. Altogether bioinformatic analysis
suggested us to screen the catalytic activity of ARti-2 in vitro
(generated using NADP /glucose/GDH) in buffer of different
pH values for
conversion to product 8 was determined by analysing
5 h under anoxic conditions and the
1
H
starting with the substrates accepted by 17β-HSDcl, T HNR
NMR in acetone-d of the crude reaction mixture. It showed
4
6
and/or T
3
HNR.
maximum conversions of 93% at pH 6.5 and 90% at pH 7.0
(Fig. 2b). However, considering the little difference in the
conversions, we performed most of the transformation using
potassium phosphate buffer of pH 7.0.
We planned to investigate the kinetic profile of ARti-2
using 10a/10b as a substrate which is formed in situ by the
The codon-optimized gene of ARti-2 cloned into the pET-
1
9b vector was overexpressed in E. coli BL21(DE3) and the
protein was purified using Ni–NTA affinity chromatography
see the ESI,† page S6). The catalytic activity of the purified
ARti-2 was tested using emodin (9) as a substrate. For this, 9
was incubated with ARti-2 in the presence of Na
(
2
S
2
O
4
&
2 2 4
reduction of emodin (9) in the presence of Na S O under
+
NADPH
[generated
using
NADP /glucose/glucose
anoxic conditions. However, maintaining such anoxic
conditions to prevent oxidation of 10a/10b back to emodin
(9) during kinetic measurements remained a challenge. In
addition, the spectral overlap of the substrate, cofactor and
the product using the UV-vis spectrophotometric method
prevented us from determining the kinetic parameters for
ARti & other related enzymes. That is why thus far no report
has been published that investigates the kinetic profile of
enzymes catalysing the reduction of anthrols. Therefore, we
dehydrogenase (GDH)] in 50 mM KPi buffer (pH 7.0, 1 mM
EDTA, 1 mM DTT) under anoxic conditions for 14 h
2 2 4
(Scheme 1). Na S O was used for the reduction of 9 to a
tautomeric mixture of emodin hydroquinone 10a/10b in situ,
one of which acts as a substrate for the anthrol reductases.
This resulted in the formation of (R)-3,8,9,10-tetrahydroxy-6-
2
Scheme 1 Chemoenzymatic asymmetric reductive dearomatization of
emodin (9). ARti-2 catalyses the NADPH-dependent reduction of the
tautomer of emodin hydroquinone 10a/10b to 8.
Fig. 2 a) Denaturation curve of ARti-2. b) pH profile of ARti-2
determined from chemoenzymatic reduction of emodin (9) in buffer of
different pH values.
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used the reverse-phase HPLC method to analyse the ARti &
ARti-2-catalysed NADPH-dependent reduction of anthrols
1
0a/10b to (R)-8. At first, we varied the substrate
concentration and analysed the amount of (R)-8 formed to
measure the velocity. KM and Vmax (= kcat·[ET]) were
determined by plotting the reaction velocity versus substrate
concentration that is fitted to the Michaelis–Menten
equation. The catalytic efficiency (η = kcat/K ) of ARti-2 was
cat
M
3
−1
−1
−1
−1
found to be 4.01 × 10 s
M
which is higher than that of
M ) when 9 was employed as a
3
ARti (ηcat = 3.56 × 10
s
substrate in the reaction (Fig. 3). This is the first time that
the impression of the kinetics with any of the ARs for anthrol
reduction has been obtained, which provides significant
catalytic insights into such biotransformation.
To further validate the anthrol reductase activity of ARti-2,
we also used lunatin (12) and citreorosein (15) as substrates.
For this, anthraquinones 12 or 15 were incubated with ARti-2
Fig. 4 a) Chemoenzymatic reduction of lunatin (12) and citreorosein
(
15) in the presence of sodium dithionite, ARti-2 & NADPH. b and c)
Kinetic profiles of ARti-2-catalysed reduction of 13 & 16.
2 2 4
in the presence of Na S O and NADPH, which resulted in
the formation of the corresponding dihydroanthracenones 14
and 17 in 62% and 72% yields, respectively (Fig. 4a).
to reduce simple cyclic ketone 24 or 2-methyl-2-cyclohexen-1-
one (25). Likewise, emodin anthrone 26 was also not reduced
by ARti-2. Moreover, plausible reduction of anthrol tautomer
10b by ARti-2 hints towards its ene reductase activity
(Scheme 1). However, neither 25 nor menadione (27), both of
which contain an alkene bond, was reduced by ARti-2.
Altogether, substrate screening suggests that ARti-2
specifically reduces anthrols but shows little promiscuity
Again, no product was obtained when the transformations
were performed without Na S O , ruling out anthraquinones
2
2 4
1
2 and 15 to be direct substrates for ARti-2 (Fig. 4a). To
determine the kinetic profile of ARti-2-catalysed reduction of
hydroquinones lunatin (13a/13b) and citreorosein (16a/16b)
as substrates, reactions with varying anthraquinone (12 or
1
5) concentrations were performed in a closed system and
4
towards other substrates such as estrone & T HN.
analysed using HPLC to determine the reaction velocity.
Kinetic parameters were calculated from the Michaelis–
Menten plot of substrate concentration versus reaction
velocity (see the ESI,† page S15). The catalytic efficiencies
Finally, characterization of ARti-2 as an AR & its presence
in an unrelated BGC prompted us to explore its relevance to
the biogenesis of dimeric (−)-flavoskyrin (30) and (−)-
rugulosin (31), which have been isolated from fungus P.
1
8
(
K
cat/K
M
) of ARti-2-catalysed reduction with 13 and 16 were
islandicum in particular. In support, we found that T.
islandicus WF-38-12 also contains an atypical SDR (GenBank
CRG89872.1) in the BGC containing ARti-2 (see the ESI,†
2
−1
−1
3
−1
−1
found to be 6.81 × 10
respectively (Fig. 4b and c).
In addition, we tested compounds 4, 18, and 20–23 as
substrates with ARti-2, which were reduced by either T HNR
s
M
and 3.95 × 10
s
M ,
−
107
Table S1), which shows 57% sequence identity (1 × 10
4
e-value) with AgnL4 (GenBank A0A411PQN7.1) of the agnestin
BGC. The knockout experiments by Simpson and co-workers
have revealed that the deletion of the AgnL4 gene
or 17β-HSDcl using NADPH (Fig. 5). HPLC analysis shows
that ARti-2 could reduce 1,3,6,8-tetrahydroxynaphthalene (4)
to (R)-scytalone (6) & estrone (18) to 17β-estradiol (19) in a
highly stereo- and regioselective manner (Fig. 5a). This shows
the ability of ARti-2 to reduce naphthol and a keto group of
estrone (18).
9
accumulated 9 in Paecilomyces variotii. Based on that, AgnL4
has been proposed to catalyse the reduction of 9 to 7 during
However, unlike T HNR, ARti-2 could not catalyse the
4
reduction of lawsone (20), 2-hydroxyjuglone (21),
1
6,17
3
-hydroxyjuglone (22) & 2-tetralone (23).
ARti-2 also failed
4
Fig. 5 a) Reduction of T HN (4) and estrone (18) by ARti-2 in the
Fig. 3 Kinetic profiles of ARti-2 & ARti-catalysed reduction of emodin
9) in the presence of Na
presence of NADPH showcases the catalytic promiscuity of ARti-2. b)
Substrates not accepted by ARti-2.
(
2 2 4
S O .
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and a naphthol (4). For the first time, we could determine
the kinetic parameters of anthrol reductases (ARti & ARti-2)
using HPLC. Although the role of another associated gene,
CRG89872.1, could not be verified through in vitro
experiments, it might be crucial for the biosynthesis of (R)-8.
Therefore, considering the isolation of bisanthraquinones,
(
−)-flavoskyrin (30) & (−)-rugulosin (31), we have speculated
the role of CRG89872.1, ARti-2 and the non-enzymatic
transformations in their biosynthesis. As no BGC is yet
identified for these complex products, the current study may
provide useful insight into the biogenesis of this class of
dimeric natural products.
Fig. 6 Proposed role of ARti-2 in the biosynthesis of bisanthraquinoid
metabolites isolated from T. islandicus (previously P. islandicum).
Conflicts of interest
9
There are no conflicts to declare.
agnestin biosynthesis. Although the catalytic functions of
either AgnL4 or other related enzymes are not yet verified
through in vitro experiments, we aim to identify the function
of CRG89872.1. For this purpose, we tried to express
CRG89872.1 in E. coli BL21(DE3) from a codon-optimised
gene cloned into the pET-19b vector. However, despite several
attempts and applying different conditions, the CRG89872.1
gene could not be expressed. Then, we tried to express its
Acknowledgements
We are grateful to SERB-DST, New Delhi (CRG/2018/002682)
for funding this research; DBT for the fellowship to S. K. S.
and UGC for fellowships to A. R. and A. D.; and the Director,
Centre of Biomedical Research for research facilities.
9
19
analogous proteins: AgnL4, AflX (GenBank Q6UEF2.1), and
Notes and references
2
0
MdpK (GenBank C8VQ62.1) in E. coli. However, we could
not obtain the desired protein in any case & hence couldn't
verify the in vitro role of CRG89872.1.
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2
1
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1
1,23
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